It is believed that 554,740 Americans died from cancer in 1996. Ten years later, the National Cancer Institute estimated that 570,280 Americans would die of cancer annually. Existing cancer treatment technologies are clearly not sufficient. Despite notable progress, there is a continuing need for better drugs and therapeutic modalities to combat cancer and improve the quality and the duration of life of cancer patients. Of particular interest are orally active agents that possesses antitumor activity either alone or in conjunction with other chemotherapeutic or anticancer agents.
The role of vitamins in cancer prevention and treatment has been a subject of interest. Ascorbate is known to act as an adjunct in improving responses to various types of cancer therapies by potentiating growth inhibitory effects of certain agents and increasing the cytotoxicity of others. Ascorbate may even reverse malignant cell transformation. The combination of Vitamin C and Vitamin K3 has been studied as a possible potentiating therapeutic modality for conventional chemotherapy. See, for example, U.S. Pat. No. 7,091,241 (by several of the present inventors and colleagues; Taper H S et al., 1987, Int J Cancer. 40:575-9; Noto V et al., Cancer 1989, 63:901-6; Taper H S et al., Anticancer Res. 1992, 12:1651-4; De Loecker W et al., Anticancer Res. 1993, 13:103-6; Taper H S et al., 1996, Anticancer Res. 16:499-503). The present inventors have now discovered that the active tolan compounds of the present invention combined with ascorbate are useful for inhibiting cancer cell proliferation, inhibiting tumor angiogenesis, promoting tumor cell apoptosis, and thereby treating subjects with cancer.
Vitamin C/Ascorbate
The chemical structure of Vitamin C (sodium ascorbate) is shown below:
Vitamin C (abbreviated herein as “VitC or “VC”) acts as a pro-oxidant, and has been evaluated as an antitumor agent. Several in vitro studies demonstrated that VC selectively accumulated in, and was toxic to, a variety of human tumor cells in culture. These included malignant melanoma cells, leukemia cells, neuroblastoma cells, ascites tumor cells as well as acute lymphoblastic leukemia, epidermoid carcinoma and fibrosarcoma cells.
Several case reports describe favorable outcomes in cancer patients undergoing high dose intravenous VC therapy. E. Cameron and L. Pauling (Proc Natl Acad Sci USA 73:3685-9, 1976). reported the effect of administering supplemental ascorbate (10 g/day intravenously (i.v.) for 10 days followed by 10 g/day orally thereafter) to 100 terminal cancer patients as part of routine management of these patients. The “controls,” 1000 subjects matched for age and sex, were left untreated. These were individuals who suffered from cancer of the same primary organ type and histological type as the patient group. The mean survival time (MST) for ascorbate-treated subjects (>210 days) was more than 4.2 times greater than that of the controls (50 days) (p<<0.0001). Six of the 100 treated subjects had ovarian cancer. When their progress was compared to that of disease-matched controls, the MST of 148 days was twice as long as the controls (MST: 71 days; p<0.005). The results suggested that VC may be of value in the treatment of advanced ovarian cancer. Two later randomized, double-blind, placebo-controlled, clinical trials (Creagen et al., New Eng. J. Med. 301:687-690, 1979; Moertel et al., New Eng. J. Med. 312:137-141, 1985) that were designed to evaluate the effectiveness of 10 g of oral VC in patients with advanced cancer, reported no benefits of oral VC treatment. More recently, these studies have been criticized (Riordan et al., Med. Hypotheses 44:207-213, 1995) because the oral VC dose of 10 g/day is not believed to be sufficient to achieve plasma concentrations that were found to be cytotoxic for tumor cells in culture. Finally, a number of case studies (Riordan et al., supra; Riordan et al., P.R. Health Sci. J. 23:115-118, 2004; Drisko et al., J. Am. Coll. Nutr. 22:118-23, 2003) reported the effects of high i.v. doses of VC in patients with breast, colorectal, ovarian, pancreatic, renal cell carcinoma. VC doses ranged from 10 to 100 g given twice per week with the majority of doses being 60-70 g per infusion. The results of these case reports suggested that high i.v. doses of VC do not interfere with conventional anticancer therapy; are generally not toxic to cancer patients with normal renal function; and induce a small number of complete remissions. This high dose i.v. regimen of VC administration, while manifesting antitumor activity, is financially burdened an inconvenient as it requires additional doctor visits.
VC usage in humans is well-documented, and the vitamin is well tolerated in animals. Mice given daily VC doses of 6.5 g/Kg body weight for 6 weeks and 2 g/Kg for 2 years showed no abnormal rates of mortality, weight changes, blood chemistry, hematology, histology, or other pathologies (Klenner, F R, 1951, South Med J 113:101-7). This reference includes a table of therapeutic doses ranging from 35 g/day for a 220 pound man to 1.2 g/day in infants. Also indicated were maintenance doses of 60 mg/kg/day (i.e., about 2180 mg/day) and 75 mg/day for these respective groups. The only systemic toxicity noted at these doses has been diarrhea/gastrointestinal upset, in which case the doses are injected, bypassing these complications.
Vitamin K3/Naphthoquinone
Vitamin K3 (“VK3; or menadione is a polycyclic aromatic ketone, based on 1,4-naphthoquinone, with a 2-methyl substituent. Its chemical name is 2-methyl-1,4-naphthoquinone or 2-methylnaphthalene-1,4-dione, and its the chemical formula is C11H8O2, molecular mass 172.18. The chemical structure is shown below.
This synthetic derivative of vitamin K1 exhibits antitumor activity against tumor cell lines from liver, cervix, nasopharynx, colon, lung, stomach, breast, leukemia and lymphoma (Nutter et al., Biochem. Pharmacol. 41:1283-92, 1991; Wu et al., Life Sci. 52:1797-804, 1993; Wu et al., Br. J. Cancer 35:1388-1393, 1999). Vitamin K3 and its derivatives have also been employed as radiosensitizers because of their ability to concentrate selectively in malignant cells of various human tumors and metastases (including liver, kidney, bladder, prostate, stomach, intestine and colon cancers) while minimally accumulating in bone marrow (Halsall et al., Urology 1:550-2, 1973; Marrian et al., Acta radiol. Ther. Phys. Biol. 8:221-46, 1969). VK3 has also proven effective against multiple drug-resistant leukemia cell lines and adriamycin-resistant leukemia cells in rats (Nutter et al., supra; Parekh et al., Cancer Lett. 61:147-56, 1992). Weekly i.p. administration of VK3 (10 mg/2 mL) to hepatoma-bearing rats for 4 weeks increased survival to 60 days for test rats compared to 17 days for controls and resulted in 5 out of 16 long term survivors (Su et al., Gaoxiong Yi Xue Ke Xue Za Zhi 7:454-9, 1991). VK3 (150-200 mg/day i.v.) has been shown (Mitchell et al., Acta Radiol. Ther. Phys. Biol 3:329-41, 1965) to radiosensitize patients with inoperable bronchial carcinoma and to chemosensitize patients to chemotherapeutic agents. When VK3 was added to cultured human oral epidermoid carcinoma cells with another chemotherapeutic agent, synergism was observed with bleomycin, cisplatin, dicarbazine and 5-fluorouracil (5-FU) and an additive effect was observed with actinomycin D, cytarabine, doxorubicin, hydroxyurea, mercaptopurine, mitomycin C, mitoxantrone, thiotepa, vincristine and VP-16 (Su et al., Cancer Treat Rep. 71:619-25, 1987). Synergistic activity was also observed between VK3 and doxorubicin, 5-FU, and vinblastine against nasopharyngeal carcinoma cells and with doxorubicin or mitomycin against MCF-7 breast cancer cells with pretreated with VK3 (Liao et al., Int. J. Oncol. 17:323-8, 2000; Tetef et al., J. Cancer Res. Clin. Oncol. 121:103-6, 1995; Tetef et al., Invest. New Drugs 13:157-62, 1995). A study with rats (Gold et al., Cancer Treat. Rep. 70:1433-5, 1986; Lamson et al., Altern. Med. Rev. 8:303-18, 2003) showed that the combination of methotrexate (0.75 mg/kg/day) and menadione (250 mg/kg/day) resulted in 99% inhibition of tumor growth. Decreasing the dosage of VK3 to 225 mg/kg/day resulted in 84% inhibition. In addition, circulating levels of VK3 as low as 1 μM acted synergistically with methotrexate. In phase I clinical trials in humans (Akman et al., Proc. Am. Soc. Clin. Oncol. 7:76, 1988; Margolin et al., Canc Chemother. Pharmacol. 36:293-8, 1995), vitamin K3 was administered at doses of 400-500 mg/day over 3-5 consecutive days without any appreciable toxicity. When VK3 was administered in conjunction with mitomycin C, a maximum tolerated dose of VK3 (2.5 g/m2 in a 48-hour i.v. infusion) followed by mitomycin C (15 mg/m 2) every four weeks produced no hemolysis. This trial was followed by two phase II trials (Tetef et al., supra). In the first trial, 23 advanced lung cancer patients displayed a median survival of 5.5 months. Two patients had objective response lasting 3.5 to 13 months, while 26% exhibited some tumor regression. However, 30% of patients exhibited hematologic toxicity. In the second trial, 43 gastrointestinal cancer patients showed no objective response to the therapy.
Due to its fat solubility, Vitamin K1 is sequestered in the liver and has been reported to disrupt blood clotting, resulting in clot formation s and the possibility of thrombotic phenomenon (Suttie, J W, “Vitamin K”, In: Handbook of Vitamins: Nutritional, Biochemical and Clinical Aspects, L J Machlin, ed., Marcel Dekker, Inc., New York, Chap. 4, pp. 147-198, 1984). VK3, is water soluble in the bisulfate form and does not appear to accumulate in appreciable amounts in the liver. Some of the present inventors and colleagues (Jamison, J. et al., J. Nutr. 131:158 S-60S, 2001) found no hepatotoxicity in livers of nude mice given appreciably larger doses of VK3 and no effects on bone marrow or blood clotting. A long term toxicity study in CH3 inbred rats showed no appreciable toxicities in any of the animals.
The LD50 of VK3 in mice is 500 mg/Kg. No mortality was observed in mice given oral doses of 200 mg/Kg. In the same (Molitor et al., Proc. Soc. Exp. Biol. Med. 43:125-8, 1940) study, chronic toxicity was evaluated for oral doses of K3-250, 350 or 500 mg/Kg administered daily over 30 days. The 500 mg/Kg dose was toxic. The 350 mg/Kg dose produced a marked drop in erythrocyte count and hemoglobin. The 250 mg/Kg dose did not affect either parameter or the animals' growth curves. Furthermore, in phase I clinical trials in humans (Akman et al., supra), vitamin K3 has been administered at doses of 400-500 mg/day over 3-5 consecutive days without any appreciable toxicity. VK3 did not produce toxicity in humans even with protracted administration at these doses. Phase I and Phase II clinical trials have been performed (Tetef et al., 1995, supra; Klenner, F. R., In: Physician's Handbook on Orthomolecular Medicine, 3rd ed, R J Williams et al., eds., Pergamon, New York, pp. 51-59, 1977) using VK3 in combination with mitomycin C (a drug which is far more toxic than VC) for lung and gastrointestinal cancers. In these studies, VK3 was well tolerated even though it was administered i.v., generally a more toxic route (Tetef et al., supra.
Studies of Combined Treatment with VC and VK3 
VC was shown to accumulate in tumors, and could reverse malignant cell transformation and exert cytotoxic effects on tumor cells. VC given alone required high doses to achieve inhibitory effects. VK3 inhibited growth of mammalian tumor cells in a culture, and required high dosages to achieve a desirous effect when administered alone (Noto, V et al. 1989, Cancer 63:901-6)
VC at 1 g/Kg and VK3 at 10 mg/Kg were injected into mice bearing ascitic liver tumor (TLT) before or after a single treatment of several cytotoxic drugs and the effects on survival (Taper, H S et al., 1987, Int. J. Cancer 40:575-9). Combined i.p. administration of these vitamins produced a distinct chemotherapy-potentiating effect for all drugs examined, especially when injected before chemotherapy. This potentiating treatment did not increase the general and organ toxicity that accompanies cancer chemotherapy.
The use of a Vitamin C/Vitamin K3 combination in conjunction with radiotherapy to treat cancer was studied (Taper, H S et al., 1996, Anticancer Res 16:499-504). The effect of intraperitoneal and oral pretreatment with combined VC and VK3 on the single dose radiotherapy of a transplantable solid mouse tumor was investigated. Groups of mice bearing intramuscularly transplanted liver tumors, were orally and parenterally pretreated with combined VC and VK3 and locally irradiated with single doses of 20, 30, or 40 Gy of X-rays. Tumor dimensions were measured twice weekly and the approximate tumor volumes in treatment groups were compared. This nontoxic pretreatment produced significant potentiation of radiotherapy induced by 20 to 40 Gy of X-rays. Combined VC with VK3 was believed to constitute a redox-cycling system producing peroxide and other reactive oxygen species (ROS) to which cancer cells are selectively sensitive.
Administration of Vitamin C and Vitamin K3 prior to treatment with certain chemotherapeutic agents was disclosed in Taper, H et al., 1992, Oncology (Life Sci Adv.) 11:19-25)
IP injection of Vitamin C and Vitamin K3 was given as a pretreatment to increase tumor sensitization to the action of Oncovin (Taper, H S et al., 1992, Anticancer Res 12:1651-4). Combined VC and VK3 therapy given i.p. 3 hours before i.p. administration of single dose of oncovin to which murine ascites liver tumor (T.L.T.) was completely resistant, was investigated. This pretreatment sensitized the tumor resistant to oncovin, whereas a separate pretreatment with either VC or VK3 alone had no effect. This tumor sensitization to chemotherapy was completely suppressed by catalase pretreatment, suggesting that hydrogen peroxide generation with subsequent oxidative stress and its consequences may be involved. This sensitization was without any increased general and organ toxicity, making it a potentially addition to classical protocols of human cancer chemotherapy.
The growth inhibitory effects of VC and VK3 combined with various chemotherapeutic agents was tested in vitro on cultured human endometrial adenocarcinoma (AN3CA) cells De Loecker, W. et al., 1993, Anticancer Res 13:103-6. In well defined conditions of cell confluence and at the dose levels applied, a synergistic effect on growth inhibition was observed. The combined vitamins when reaching their own synergistic cytotoxicity levels frequently obscure the additional synergistic effects attributable to the chemotherapeutic agents. The formation of ROS radicals during treatment, possibly accentuated by less defined secondary mechanisms, appeared to be responsible for the observed stimulated cytotoxicity.
Action of Polyphenolic Compounds
Uncontrolled imbalance between cell proliferation and cell differentiation or cell death may result in the development of malignant or cancerous clones of cells which are commonly less differentiated than their normal counterparts. Thus, promising targets for cancer intervention are induction of (i) differentiation of pre-malignant or malignant cells into more normal cells and (ii) tumor-specific cell death during the process of carcinogenesis or tumor development. Compounds which induce differentiation or cell death are candidates for cancer chemoprevention and/or chemotherapy (Hong W K and Sporn M B, Science, 1997, 278:1073-7; Suh N. et al., Anticancer Res., 1995, 15:233-9; Fimognari C. et al., Biochem Pharmacol., 2004, 68:1133-8). In the last several years, hundreds of plant extracts have been evaluated for their potential as cancer chemopreventive agents and for their ability to induce cell death (Clement M. V. et al., Blood, 1998, 92:996-1002; Cooke D. et al., Eur J Canc., 2005, 41:1931-40).
Many of these compounds inhibit the cellular events associated with all 3 stages of carcinogenesis (initiation, promotion and progression). One strategy employs phenolic compounds that prevent or attenuate cancer formation by blocking one or several steps in this multistage process. A non-flavonoid polyphenol, resveratrol (3,5,4′-trihydroxy-trans-stilbene, depicted below), is a typical example of such a compound.
Its as diverse bioactivities (Aggarwal B B et al., Anticancer Res., 2004, 24:2783-2840; Fremont L., Life Sci., 2000, 66:663-73) include antioxidant activity, modulation of lipid and lipoprotein metabolism, anti-platelet aggregation, vasorelaxing activity, anticancer activity, estrogenic activity. A variety of mechanisms of action for this compound have been suggested or reported including: increased levels of cell death, activation of phase II detoxification and decreased levels of cell division, DNA synthesis and inflammation (Aggarwal et al., supra; Aziz M H et al., Int J Oncol., 2003 23:17-28; Dong Z., Mutat Res., 2003, 523-524:145-50; Fremont L., supra; Gusman J. et al., Carcinogenesis, 2001, 22:1111-7; Jang M. et al., Science, 1997, 275:218-20; Savouret J. F. et al., Biomed Pharmacother., 2002, 56:84-7; Signorelli P. et al., J Nutr Biochem., 2005, 16:449-66)
Resveratrol was reported to inhibit cell proliferation and cause apoptotic cell death by modulating numerous key mediators of cell cycle and survival signaling. Depending on the concentrations, resveratrol “switched” cells between reversible cell cycle arrest and irreversible apoptosis. Specifically, resveratrol treatment blocked the cell cycle in the G0/G1, G1/S transition, S phase or G2/M phases by suppressing cyclins and their corresponding kinases, by increasing p53 levels or by inhibiting DNA synthesis. It also causes up-regulation of pro-apoptotic members of the Bcl-2 family and down-regulation of anti-apoptotic members of this family. Finally, resveratrol inhibits NF-κB and AP-1 signaling pathways, their upstream kinases and their downstream targets (including inducible cyclooxygenase-2, inducible nitric oxide synthase and matrix metallo-protease-9. Thus, resveratrol inhibits proliferation and induces cell death. (See references cited supra).
Resveratrol is considered a phytoestrogen because of its structural homology to the estrogens and it can compete with estrogens for their receptors and activate hormone receptor-mediated gene transcription. However, it can also exert an anti-estrogen action and inhibit hormone-induced carcinogenesis with the agonistic or antagonistic hormonal activity depending on the intake concentration, tissue-specific expression of estrogen receptors, cofactors present for DNA binding and different gene promoters (Aggarwal et al., supra; Fremont, supra). Likewise, resveratrol represses transcription or translation of different classes of androgen up-regulated genes via a reduction in androgen receptor (AR) content (Mitchell S H et al., Cancer Res, 1999, 59:5892).
While the antitumor mechanisms of resveratrol are pleiotropic, and it appears to be a promising antitumor agent in part because it affects the 3 stages of carcinogenesis, its use has been hampered by its relatively low aqueous solubility and its apparent lack of specificity to tumor cells. Resveratrol was also significantly toxic to normal cells (Aggarwal et al., supra). The present invention is directed to a distinct class of diphenyl compounds, the tolans, and their utility as anticancer agents with advantageous properties compared to resveratrol.
U.S. Pat. Nos. 6,599,945 and 7,094,809 (co-invented by one of the present inventors) disclose several hydroxytolan compounds and their uses in methods of inhibiting the formation of infectious herpes virus particles or for treating gonorrhea caused by Neisseria gonorrhoeae. U.S. Pat. Nos. 6,197,834 and 6,355,692 disclose certain hydroxylated stilbenes, and specifically resveratrol, for similar uses. The use of resveratrol in suppressing or treating cancer is also disclosed in U.S. Pat. No. 6,008,260.